Antisense modulation of complement component C3 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of complement component C3. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding complement component C3. Methods of using these compounds for modulation of complement component C3 expression and for treatment of diseases associated with expression of complement component C3 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of complement component C3. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding complement component C3. Such compounds have been shown to modulate the expression of complement component C3.

BACKGROUND OF THE INVENTION

[0002] The complement system provides a rapid and efficient means of protecting a host from invasive microorganisms. The complement system consists of about 30 proteins, acting within a cascade-like reaction sequence, that serve as control proteins or as cellular receptors. The complement system can be activated by any of three pathways, either the antibody-dependent classical pathway, the alternative pathway, or the mannan-binding lectin (MBL)/MBL-associated serine protease pathway. Following activation, the complement components form membrane attack complexes which elicit a number of biological effects such as chemotaxis of leukocytes, degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction, and the increase of vascular permeability (Kirschfink, Immunopharmacology, 1997, 38, 51-62).

[0003] Due to its diverse biological activities, complement is a key mediator of inflammation, a natural response to the host tissue response to any injury. There is also increasing evidence that complement significantly contributes to the regulation of the immune response. Inappropriate or excessive activation of the complement system can lead to harmful, potentially life-threatening consequences due to severe inflammatory tissue destruction. These consequences are clinically manifested in various disorders including septic shock, multiple organ failure, hyperacute organ failure, autoimmune disorders, and CNS inflammation (Kirschfink, Immunopharmacology, 1997, 38, 51-62).

[0004] The role of complement component C3 (also known as C3) is indispensable because it functions in all three pathways in complement activation. The physiological activities of complement component C3 include opsonization and cellular activation via ligation of complement receptors CR1, CR2, and CR3; anaphylatoxic activities mediated by C3a; and binding to Factor B to form the alternative pathway C3bBb C3 convertase enzyme and participation in the classical and alternative pathway with C4 convertase enzymes, C4b2a3b and C3bBbC3b. These different activities of C3 are mediated by different regions of the polypeptide and by attached carbohydrate residues (Fong et al., Genomics, 1990, 7, 579-586).

[0005] Complement component C3 was isolated and cloned from human liver (de Bruijn and Fey, Proc. Natl. Acad. Sci. U. S. A., 1985, 82, 708-712) and mapped to chromosome 19p13.3-p13.2 (Whitehead et al., Proc. Natl. Acad. Sci. U. S. A., 1982, 79, 5021-5025).

[0006] Nucleic acid sequences encoding human complement component C3 are disclosed in PCT publication WO 97/32981 (Farries and Harrison, 1997). Disclosed and claimed in U.S. Pat. No. 6,221,657 is a DNA sequence encoding a modified human complement component C3 and a vector comprising said sequence (Harrison and Farries, 2001).

[0007] The complete gene is 41 kb and consists of 41 exons. The protein is produced as a single polypeptide of approximately 200 kDa, which is then proteolytically processed to yield the mature protein. The mature protein consists of two disulfide-linked subunits, α and β, of 100 and 75 kDa, respectively (Fong et al., Genomics, 1990, 7, 579-586). While the primary site of complement component C3 synthesis is the liver, extra-hepatic synthesis is common and a number of cell types such as macrophages, keratinocytes, kidney tubular epithelial cells, and endothelial cells (Carroll, Annu. Rev. Immunol., 1998, 16, 545-568). Adipocytes are also an important source for complement component C3 (Yudkin, Eur. Heart J., 2000, 21, 1036-1039).

[0008] Muscari et al. demonstrated strong associations between serum levels of complement component C3 and a history of myocardial infarction and stroke. They also report multivariate associations between serum complement component C3 concentrations and those of insulin, triglyceride, and high-density lipoprotein cholesterol (inversely), as well as high blood pressure and obesity (Muscari et al., Eur. Heart J., 2000, 21, 1081-1090).

[0009] Nataf et al. used complement component C3 knockout mice to study the mechanisms leading to CNS inflammation and mylein destruction in multiple sclerosis in its animal model, experimental allergic encephalomyelitis (EAE). Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces both wild-type and the knockout animals, there was little infiltration of the parenchyma by macrophages and T-cells. In addition, the knockout mice were protected from demyelination. These results suggest that complement component C3 might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS (Nataf et al., J. Immunol., 2000, 165, 5867-5873).

[0010] Mabbott et al. showed that a temporary depletion of complement component C3 significantly delays the onset of scrapie in mice. Transmissable spongiform encephalopathies, like scrapie, require host prion proteins for replication. Depletion of complement component C3 reduces the early accumulation of detergent insoluble, proteinase-resistant prion proteins on the follicular dendritic cells (Mabbott et al., Nat. Med., 2001, 7, 485-487).

[0011] The pharmacological modulation of complement component C3 expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions.

[0012] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of complement component C3.

[0013] Anti-complement component C3 antibodies have been used to block the complement cascade (Kirschfink, Immunopharmacology, 1997, 38, 51-62).

[0014] To date, investigative strategies aimed at modulating C3 function have involved the use of antibodies and gene knockouts in mice.

[0015] Consequently, there remains a long felt need for additional agents capable of inhibiting complement component C3 function.

[0016] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of complement component C3 expression.

[0017] The present in invention provides compositions and methods for modulation complement component C3 expression.

SUMMARY OF THE INVENTION

[0018] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding complement component C3, and which modulate the expression of complement component C3. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of complement component C3 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of complement component C3 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding complement component C3, ultimately modulating the amount of complement component C3 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding complement component C3. As used herein, the terms “target nucleic acid” and “nucleic acid encoding complement component C3” encompass DNA encoding complement component C3, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of complement component C3. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0020] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding complement component C3. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding complement component C3, regardless of the sequence(s) of such codons.

[0021] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′or 3′) from a translation termination codon.

[0022] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0023] Although some eukaryotic MRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0024] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0025] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0026] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.

[0027] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0028] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0029] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0030] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0031] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0032] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0033] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0034] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0035] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0036] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0037] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0038] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0039] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0040] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0041] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0042] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂-O—CH₂-N(CH₂)₂, also described in examples hereinbelow.

[0043] A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0044] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂-CH═CH₂) and 2′-fluoro (2′-F) The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0045] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡—C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0046] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0047] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0048] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0049] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0050] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0051] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0052] The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0053] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0054] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0055] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0056] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0057] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0058] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of complement component C3 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0059] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding complement component C3, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding complement component C3 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of complement component C3 in a sample may also be prepared.

[0060] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0061] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0062] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.

[0063] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0064] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0065] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0066] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0067] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0068] Emulsions

[0069] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.

[0070] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0071] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0072] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0073] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0074] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0075] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0076] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0077] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0078] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0079] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0080] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0081] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0082] Liposomes

[0083] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0084] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0085] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0086] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0087] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0088] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0089] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0090] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0091] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0092] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0093] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0094] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0095] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0096] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci. 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0097] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0098] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0099] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0100] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0101] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0102] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0103] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0104] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0105] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0106] Penetration Enhancers

[0107] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0108] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0109] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0110] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0111] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, N.Y., 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0112] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0113] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0114] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0115] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0116] Carriers

[0117] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0118] Excipients

[0119] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0120] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0121] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0122] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0123] Other Components

[0124] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0125] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0126] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0127] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0128] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0129] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites

[0130] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

[0131] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

[0132] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a 2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

[0133] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

[0134] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

[0135] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

[0136] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

2,2′-Anhydrol[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0137] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

[0138] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0139] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃CN (200 mL). The residue was dissolved in CHCL₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0140] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0141] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0142] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0143] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0144] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0145] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0146] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0147] In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160 ° C. was reached and then maintained for 16 h (pressure <100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0148] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0149] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH₂Cl₂ and the combined organic phase was washed with water, brine and dried over anhydrous Na₂SO₄. The solution was concentrated to get 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0150] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1 M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and evaporated to dryness. The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH₂Cl₂ to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0151] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0152] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0153] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P₂O₅ under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

[0154] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0155] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4,-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0156] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂-O—CH₂-N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0157] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O²-,2-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl uridine

[0158] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃ solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine) gives the title compound.

5 ′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0159] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.

Example 2

[0160] Oligonucleotide Synthesis

[0161] Unsubstituted and substituted phosphodiester (P=O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

[0162] Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.

[0163] Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0164] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0165] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or U.S. Pat. No. 5,625,050, herein incorporated by reference.

[0166] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0167] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0168] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0169] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0170] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

[0171] Oligonucleoside Synthesis

[0172] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleo-sides, as well as mixed backbone compounds having, for instance, alternating MMI and P=O or P=S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0173] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0174] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0175] PNA Synthesis

[0176] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

[0177] Synthesis of Chimeric Oligonucleotides

[0178] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

[0179] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligo-nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0180] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxy-ethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0181] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxy phosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0182] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0183] Oligonucleotide Isolation

[0184] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0185] Oligonucleotide Synthesis—96 Well Plate Format

[0186] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta--cyanoethyldiisopropyl phosphoramidites.

[0187] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0188] Oligonucleotide Analysis-96 Well Plate Format

[0189] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0190] Cell Culture and Oligonucleotide Treatment

[0191] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 6 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR.

[0192] T-24 Cells:

[0193] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0194] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0195] A549 Cells:

[0196] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0197] NHDF Cells:

[0198] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0199] HEK Cells:

[0200] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0201] HepG2 Cells:

[0202] The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, Va.). HepG2 cells were routinely cultured in Eagle's MEM supplemented with 10% fetal calf serum, non-essential amino acids, and 1 mM sodium pyruvate (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0203] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0204] HEPA 1-6 Cells:

[0205] The mouse hepatoma cell line HEPA 1-6 is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse and is supplied by the American Type Culture Collection (Manassas, Va.). The cells are propagated in Dulbecco's minimal essential medium with 10% fetal bovine serum. Cells are subcultured by removing the medium, adding fresh 0.25% trypsin, 0.03% EDTA solution and letting the culture sit at room temperature for 3 minutes. Trypsin is then removed and the culture allowed to sit an additional 5 minutes until the cells begin to detach, at which point, fresh medium is added.

[0206] Treatment with Antisense Compounds:

[0207] When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0208] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.

Example 10

[0209] Analysis of Oligonucleotide Inhibition of Complement Component C3 Expression

[0210] Antisense modulation of complement component C3 expression can be assayed in a variety of ways known in the art. For example, complement component C3 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0211] Protein levels of complement component C3 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to complement component C3 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0212] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0213] Poly(A)+ mRNA Isolation

[0214] Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0215] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

[0216] Total RNA Isolation

[0217] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

[0218] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0219] Real-time Quantitative PCR Analysis of Complement Component C3 mRNA Levels

[0220] Quantitation of complement component C3 MRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0221] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0222] PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0223] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, Analytical Biochemistry, 1998, 265, 368-374.

[0224] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0225] Probes and primers to human complement component C3 were designed to hybridize to a human complement component C3 sequence, using published sequence information (GenBank accession number K02765, incorporated herein as SEQ ID NO:3). For human complement component C3 the PCR primers were: forward primer: CGTGATACACCAAGAAATGATTGG (SEQ ID NO: 4) reverse primer: CTGCAGCGAGATGAGAACAAAG (SEQ ID NO: 5) and the PCR probe was: FAM-ACAACGAGAAAGACATGGCCCTCACG-TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:7) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0226] Probes and primers to mouse complement component C3 were designed to hybridize to a mouse complement component C3 sequence, using published sequence information (GenBank accession number J00367, incorporated herein as SEQ ID NO:10). For mouse complement component C3 the PCR primers were: forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:11) reverse primer: CGACTCCGGGCTCACAAG (SEQ ID NO: 12) and the PCR probe was: FAM-TAGCCGGACATTCAGGTTGATCTTCTCCT-TAMRA (SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For mouse GAPDH the PCR primers were: forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:14) reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:15) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

[0227] Northern Blot Analysis of Complement Component C3 mRNA Levels

[0228] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0229] To detect human complement component C3, a human complement component C3 specific probe was prepared by PCR using the forward primer CGTGATACACCAAGAAATGATTGG (SEQ ID NO: 4) and the reverse primer CTGCAGCGAGATGAGAACAAAG (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0230] To detect mouse complement component C3, a mouse complement component C3 specific probe was prepared by PCR using the forward primer AAGCTGTGCCACAGTGAAATGT (SEQ ID NO: 11) and the reverse primer CGACTCCGGGCTCACAAG (SEQ ID NO: 12). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0231] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0232] Antisense inhibition of human complement component C3 expression by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human complement component C3 RNA, using published sequences (GenBank accession number K02765, incorporated herein as SEQ ID NO: 3 and GenBank accession number M55658, incorporated herein as SEQ ID NO: 17). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human complement component C3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N. D.” indicates “no data”. TABLE 1 Inhibition of human complement component C3 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TAR- GET SEQ TAR- % SEQ ID GET IN- ID ISIS # REGION NO SITE SEQUENCE HIB NO Start 3 45 ccatggtgctgggacagtgc 44 19 139964 Codon Start 3 54 aggtgggtcccatggtgctg 26 20 139965 Codon 139966 Coding 3 84 ttagtagcaggagcagcagg 25 21 139967 5′UTR 17 179 ctgttggacccactttgtag 11 22 139968 Coding 3 185 gtcgtgggcctccagcacca 76 23 139969 Coding 3 214 gtaacagtgactggaacatc 58 24 139970 5′UTR 17 231 ctgccctggactctcccagg 9 25 139971 Coding 3 254 actggacagcactagttttt 68 26 139972 5′UTR 17 257 cccagcagcatgaatgcagc 2 27 139973 5′UTR 17 271 ctgagggcatgttccccagc 16 28 139974 Coding 3 330 tgaactccctgttggctggg 79 29 139975 Coding 3 445 tctgtctggatgaagaggta 61 30 139976 Coding 3 450 tcttgtctgtctggatgaag 69 31 139977 Coding 3 455 gatggtcttgtctgtctgga 56 32 139978 Coding 3 460 gtgtagatggtcttgtctgt 20 33 139979 Coding 3 505 ttgtggttgacggtgaagat 54 34 139980 Coding 3 725 cacgtactccttcacctcaa 6 35 139981 Coding 3 766 aatttctctgtaggctccac 1 36 139982 Coding 3 822 agaggaacctggcggtgatg 32 37 139983 Coding 3 877 tcgccatcctggatcccgaa 40 38 139984 Coding 3 922 tcaatcggaatgcgcttgag 26 39 139985 Coding 3 1009 gacttccccaccaggtcttc 7 40 139986 Coding 3 1064 tgcctgcaccatgtcactgc 47 41 139987 Coding 3 1117 ttggtgaagtggatctggta 60 42 139988 Coding 3 1123 ggtgtcttggtgaagtggat 75 43 139989 Coding 3 1163 caccatgaggtcaaagggca 63 44 139990 Coding 3 1170 tcacgaacaccatgaggtca 31 45 139991 Coding 3 1193 ggctggagagccatcagggt 41 46 139992 Coding 3 1344 ctgcctccgagagctcctgc 68 47 139993 Coding 3 1402 ttgttggagttgcccacggt 21 48 139994 Coding 3 1430 tgtacgtagcactgagagat 25 49 139995 Coding 3 1583 caggtcctggccgggctctc 66 50 139996 Coding 3 1652 cagcgtgtagtacgccacca 49 51 139997 Coding 3 1860 tattcagcacgaacacgccc 43 52 139998 Coding 3 2246 ccgcagctctgtgatgtagt 29 53 139999 Coding 3 2279 caggcccaggtggctggccc 26 54 140000 Coding 3 2406 tcgtagagattccatttttc 45 55 140001 Coding 3 2448 cccacgtggtgatggagtct 56 56 140002 Coding 3 2483 ccctttcttgtccgacatgc 66 57 140003 Coding 3 2492 cacacagatccctttcttgt 18 58 140004 Coding 3 2499 ggtctgccacacagatccct 49 59 140005 Coding 3 2768 ttcctgcaggccggtcttta 53 60 140006 Coding 3 2788 acggcagccttgacttccac 61 61 140007 Coding 3 2953 acttggtcactgaggtctgc 57 62 140008 Coding 3 2987 caggagaattctggtctcag 15 63 140009 Coding 3 2992 ccttgcaggagaattctggt 48 64 140010 Coding 3 2997 gggtcccttgcaggagaatt 45 65 140011 Coding 3 3127 tccaggtaatgcacagcgat 48 66 140012 Coding 3 3214 gccagctgctgggtgtaccc 74 67 140013 Coding 3 3219 tgaaggccagctgctgggtg 50 68 140014 Coding 3 3298 aagaccttgaccacgtaggc 42 69 140015 Coding 3 3304 agagagaagaccttgaccac 34 70 440016 Coding 3 3330 agtcgatggcgatgaggttg 66 71 440017 Coding 3 3383 gggcttctgcttctccagga 59 72 440018 Coding 3 3395 gaagaccccgtcgggcttct 37 73 140019 Coding 3 3425 ttcttggtgtatcacgggcg 60 74 140020 Coding 3 3436 ccaccaatcatttcttggtg 44 75 140021 Coding 3 3517 tcgcaaatatctttagcctc 0 76 140022 Coding 3 3533 gctgttgacctgctcctcgc 0 77 140023 Coding 3 3606 cagtgtaggatctctgtagg 52 78 140024 Coding 3 3692 cttatctttggctgtggtca 42 79 140025 Coding 3 3714 taccagggtcctcccagcgg 45 80 140026 Coding 3 3748 gcataggatgtggcctccac 27 81 140027 Coding 3 3871 tggaacaccatgaaggtggc 3 82 140028 Coding 3 3887 gtattgagccaaggcttgga 46 83 140029 Coding 3 3965 ggtgatcttggagctgcggc 47 84 140030 Coding 3 4187 gttcttggcatcctgaggcc 55 85 140031 Coding 3 4276 gcaaagccagtcatcatgga 61 86 140032 Coding 3 4281 ctggagcaaagccagtcatc 14 87 140033 Coding 3 4286 tgtgtctggagcaaagccag 24 88 140034 Coding 3 4355 gaaggctttgtccagctcat 79 89 140035 Coding 3 4383 ggtagatgatgagggtgttc 60 90 140036 Coding 3 4406 ctcagagtgtgagaccttgt 75 91 140037 Coding 3 4533 ccttttccggatggtagaac 48 92 140038 Coding 3 4724 gtactcgtcaaagtcattgg 59 93 140039 Coding 3 4856 ccacatgaggtagtgtttct 35 94 140040 Coding 3 4870 tcggaggagagaccccacat 28 95 140041 Coding 3 4939 tcaggccagtgctccaccca 66 96

[0233] As shown in Table 1, SEQ ID NOs 19, 20, 21, 23, 24, 26, 29, 30, 31, 32, 34, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 49, 40, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 83, 84, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 100 and 107 demonstrated at least 25% inhibition of human complement component C3 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 16

[0234] Anitsense Inhibition of Mouse Complement Component C3 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap.

[0235] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse complement component C3 RNA, using published sequences (GenBank accession number J00367, incorporated herein as SEQ ID NO: 10, GenBank accession number K02782, incorporated herein as SEQ ID NO: 111, and GenBank accession number Z37998, incorporated herein as SEQ ID NO: 112). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse complement component C3 MRNA levels by quatitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”. TABLE 2 Inhibition of mouse complement component C3 mRNA levels by chimeric phosphorothioate oligonucleo- tides having 2′-MOE wings and a deoxy gap TAR- GET SEQ TAR- % SEQ ID GET IN- ID ISIS # REGION NO SITE SEQUENCE HIB NO 139975 Exon 10 1973 tctgtctggatgaagaggta 59 30 139976 Exon 10 1978 tcttgtctgtctggatgaag 79 31 139977 Exon 10 1983 gatggtcttgtctgtctgga 76 32 139978 Exon 10 1988 gtgtagatggtcttgtctgt 52 33 139987 Exon 10 2645 ttggtgaagtggatctggta 61 42 139988 Exon 10 2651 ggtgtcttggtgaagtggat 69 43 140000 Exon 10 3931 tcgtagagattccatttttC 65 55 140012 Exon 10 4739 gccagctgctgggtgtaccc 31 67 140016 Exon 11 2377 agtcgatggcgatgaggttg 65 71 140020 Exon 10 4961 ccaccaatcatttcttggtg 71 75 140035 Exon 10 5908 ggtagatgatgagggtgttc 0 90 140044 Start 111 48 gctggtcccatagtgaagga 56 113 Codon 140045 Coding 111 79 cagcagtagcactagtagct 79 114 140046 Coding 111 95 gggagctggccaacagcagc 68 115 140047 Coding 111 276 gctcctgtcaacactgtctt 71 116 140048 Coding 111 320 tactggctggaatcttgatg 74 117 140049 Coding 111 380 ccccgaagtttgccaccact 87 118 140050 Coding 111 535 gatgacgactgtcttgccca 4 119 140051 Coding 111 730 gggcagcacgtattccttca 62 120 140052 Coding 111 895 ggccagagaaatcttcttat 57 121 140053 Coding 111 1079 ggatcccactgcgctctgcc 15 122 140054 Coding 111 1128 aatttgggtgtcttggtgaa 57 123 140055 Coding 111 1389 ttgtgcatagtgctgtaggg 63 124 140056 Coding 111 1470 cgcaggtggaagttgacatt 77 125 140057 Coding 111 1534 cttccccttgttcataacca 54 126 140058 Coding 111 1645 ggtgtagtaagccaccaggc 68 127 140059 Coding 111 1834 cttgtccacagccactagcc 32 128 140060 Coding 111 1937 agttcttcccactgcctggg 80 129 140061 Coding 111 2094 gtgtactgaccagctttgtc 74 130 140062 Coding 111 2191 ctcgccctgggtgatgaggc 43 131 140063 Coding 111 2286 tcactcctggccaggcccag 80 132 140064 Coding 111 2341 tgggaagtggcttctagaga 47 133 140065 Coding 111 2373 ttcaactcttctatggtcca 10 134 140066 Coding 111 2432 tggaatctttgagaaagatg 62 135 140067 Coding 111 2442 caggtggtgatggaatcttt 62 136 140068 Coding 111 2465 acaagctcactgccagaatc 0 137 140069 Coding 111 2595 ttgaagagcacagctctgat 55 138 140070 Coding 111 2665 ggccatgctgcagaaggctg 37 139 140071 Coding 111 2842 tccttctggcacgaccttca 75 140 140072 Coding 111 2961 tctgtgtctggcacttggtc 62 141 140073 Coding 111 3043 gtgtttcagccgctccccgt 0 142 140074 Coding 111 3053 tcacgatcaggtgtttcagc 82 143 140075 Coding 111 3095 gtgtcatgccaatcatgttc 79 144 140076 Coding 111 3193 ccctttcttgatgagctcca 82 145 140077 Coding 111 3247 gttgaaggcagcataggcag 52 146 140078 Coding 111 3276 gctgtcagccaggtgctggg 62 147 140079 Coding 111 3376 cttctgtttctccagaatca 0 148 140080 Coding 111 3399 tcctcctgaaagacaccatc 84 149 140081 Coding 111 3496 ttcctgcagtgcgatgagga 59 150 140082 Coding 111 3525 ttgacctgcccctcacagat 75 151 140083 Coding 111 3555 cctgccttgttgatgctccc 82 152 140084 Coding 111 3598 gtatggtctctgcaggttca 58 153 140085 Coding 111 3624 agggcatacccagcaatggc 71 154 140086 Coding 111 3644 ccagtttgttcatcagggcc 0 155 140087 Coding 111 3655 gtaaggttcctccagtttgt 75 156 140088 Coding 111 3702 tcctcccagcggttccgatc 75 157 140089 Coding 111 3768 ttcagcagcagcagggccag 67 158 140090 Coding 111 3786 ggcacagagtcaaagtcttt 80 159 140091 Coding 111 3865 gaataccatgaaggtagcct 47 160 140092 Coding 111 3875 ccaaggcttggaataccatg 81 161 140093 Coding 111 3925 cacatccatgttcaagtcct 39 162 140094 Coding 111 4133 tgaccctgaggtcaaacttc 55 163 140095 Coding 111 4167 ggcttcttggctgtctcagg 72 164 140096 Coding 111 4252 gatgtccaggatggacatag 27 165 140097 Coding 111 4270 aaagccagtcatcatggaga 86 166 140098 Coding 111 4361 tcttgttggagaaggctttg 21 167 140099 Coding 111 4389 atcttttctaggtagatgat 2 168 140100 Coding 111 4479 gagtagaccttgaccgaccc 84 169 140101 Coding 111 4519 atgatagaaccgggtgcatg 32 170 140102 Coding 111 4653 ggctcacaagccttgtctag 29 171 140103 Coding 111 4753 gcctgacttgatgacctgct 21 172 140104 Coding 111 4773 cctgcctgcacctcatctga 13 173 140105 Coding 111 4858 gaggccccacatgaggtact 78 174 140106 Coding 111 4885 gggcttttctccccagaggt 62 175 140107 Coding 111 4909 cttcccaatgatgtagctgg 88 176 140108 Coding 111 4929 cagtgctccacccacgtgtc 85 177 140109 Stop 111 5037 ggctgtagtcagttgggaca 50 178 Codon 140110 3′UTR 111 5067 aaatacaactgaagctttat 77 179

[0236] As shown in Table 2, SEQ ID NOs 30, 31, 32, 33, 42, 43, 55, 71, 75, 113, 114, 115, 116, 117, 118, 120, 121, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 135, 136, 138, 140, 141, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 157, 158, 159, 160, 161, 163, 164, 166, 169, 174, 175, 176, 177, 178 and 179 demonstrated at least 43% inhibition of mouse complement component C3 expression in this experiment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 17 Western Blot Analysis of Complement Component C3 Protein Levels

[0237] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to complement component C3 is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 179 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 atgcattctg cccccaagga 20 <210> SEQ ID NO 3 <211> LENGTH: 5067 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (61)...(5052) <400> SEQUENCE: 3 ctcctcccca tcctctccct ctgtccctct gtccctctga ccctgcactg tcccagcacc 60 atg gga ccc acc tca ggt ccc agc ctg ctg ctc ctg cta cta acc cac 108 Met Gly Pro Thr Ser Gly Pro Ser Leu Leu Leu Leu Leu Leu Thr His 1 5 10 15 ctc ccc ctg gct ctg ggg agt ccc atg tac tct atc atc acc ccc aac 156 Leu Pro Leu Ala Leu Gly Ser Pro Met Tyr Ser Ile Ile Thr Pro Asn 20 25 30 atc ttg cgg ctg gag agc gag gag acc atg gtg ctg gag gcc cac gac 204 Ile Leu Arg Leu Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp 35 40 45 gcg caa ggg gat gtt cca gtc act gtt act gtc cac gac ttc cca ggc 252 Ala Gln Gly Asp Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly 50 55 60 aaa aaa cta gtg ctg tcc agt gag aag act gtg ctg acc cct gcc acc 300 Lys Lys Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr 65 70 75 80 aac cac atg ggc aac gtc acc ttc acg atc cca gcc aac agg gag ttc 348 Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe 85 90 95 aag tca gaa aag ggg cgc aac aag ttc gtg acc gtg cag gcc acc ttc 396 Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr Phe 100 105 110 ggg acc caa gtg gtg gag aag gtg gtg ctg gtc agc ctg cag agc ggg 444 Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu Gln Ser Gly 115 120 125 tac ctc ttc atc cag aca gac aag acc atc tac acc cct ggc tcc aca 492 Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr 130 135 140 gtt ctc tat cgg atc ttc acc gtc aac cac aag ctg cta ccc gtg ggc 540 Val Leu Tyr Arg Ile Phe Thr Val Asn His Lys Leu Leu Pro Val Gly 145 150 155 160 cgg acg gtc atg gtc aac att gag aac ccg gaa ggc atc ccg gtc aag 588 Arg Thr Val Met Val Asn Ile Glu Asn Pro Glu Gly Ile Pro Val Lys 165 170 175 cag gac tcc ttg tct tct cag aac cag ctt ggc gtc ttg ccc ttg tct 636 Gln Asp Ser Leu Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser 180 185 190 tgg gac att ccg gaa ctc gtc aac atg ggc cag tgg aag atc cga gcc 684 Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 195 200 205 tac tat gaa aac tca cca cag cag gtc ttc tcc act gag ttt gag gtg 732 Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val 210 215 220 aag gag tac gtg ctg ccc agt ttc gag gtc ata gtg gag cct aca gag 780 Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro Thr Glu 225 230 235 240 aaa ttc tac tac atc tat aac gag aag ggc ctg gag gtc acc atc acc 828 Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu Val Thr Ile Thr 245 250 255 gcc agg ttc ctc tac ggg aag aaa gtg gag gga act gcc ttt gtc atc 876 Ala Arg Phe Leu Tyr Gly Lys Lys Val Glu Gly Thr Ala Phe Val Ile 260 265 270 ttc ggg atc cag gat ggc gaa cag agg att tcc ctg cct gaa tcc ctc 924 Phe Gly Ile Gln Asp Gly Glu Gln Arg Ile Ser Leu Pro Glu Ser Leu 275 280 285 aag cgc att ccg att gag gat ggc tcg ggg gag gtt gtg ctg agc cgg 972 Lys Arg Ile Pro Ile Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg 290 295 300 aag gta ctg ctg gac ggg gtg cag aac ctc cga gca gaa gac ctg gtg 1020 Lys Val Leu Leu Asp Gly Val Gln Asn Leu Arg Ala Glu Asp Leu Val 305 310 315 320 ggg aag tct ttg tac gtg tct gcc acc gtc atc ttg cac tca ggc agt 1068 Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser 325 330 335 gac atg gtg cag gca gag cgc agc ggg atc ccc atc gtg acc tct ccc 1116 Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 340 345 350 tac cag atc cac ttc acc aag aca ccc aag tac ttc aaa cca gga atg 1164 Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro Gly Met 355 360 365 ccc ttt gac ctc atg gtg ttc gtg acg aac cct gat ggc tct cca gcc 1212 Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 370 375 380 tac cga gtc ccc gtg gca gtc cag ggc gag gac act gtg cag tct cta 1260 Tyr Arg Val Pro Val Ala Val Gln Gly Glu Asp Thr Val Gln Ser Leu 385 390 395 400 acc cag gga gat ggc gtg gcc aaa ctc agc atc aac aca cac ccc agc 1308 Thr Gln Gly Asp Gly Val Ala Lys Leu Ser Ile Asn Thr His Pro Ser 405 410 415 cag aag ccc ttg agc atc acg gtg cgc acg aag aag cag gag ctc tcg 1356 Gln Lys Pro Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 420 425 430 gag gca gag cag gct acc agg acc atg cag gct ctg ccc tac agc acc 1404 Glu Ala Glu Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr 435 440 445 gtg ggc aac tcc aac aat tac ctg cat ctc tca gtg cta cgt aca gag 1452 Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu 450 455 460 ctc aga ccc ggg gag acc ctc aac gtc aac ttc ctc ctg cga atg gac 1500 Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg Met Asp 465 470 475 480 cgc gcc cac gag gcc aag atc cgc tac tac acc tac ctg atc atg aac 1548 Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Ile Met Asn 485 490 495 aag ggc agg ctg ttg aag gcg gga cgc cag gtg cga gag ccc ggc cag 1596 Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln 500 505 510 gac ctg gtg gtg ctg ccc ctg tcc atc acc acc gac ttc atc cct tcc 1644 Asp Leu Val Val Leu Pro Leu Ser Ile Thr Thr Asp Phe Ile Pro Ser 515 520 525 ttc cgc ctg gtg gcg tac tac acg ctg atc ggt gcc agc ggc cag agg 1692 Phe Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg 530 535 540 gag gtg gtg gcc gac tcc gtg tgg gtg gac gtc aag gac tcc tgc gtg 1740 Glu Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Val 545 550 555 560 ggc tcg ctg gtg gta aaa agc ggc cag tca gaa gac cgg cag cct gta 1788 Gly Ser Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val 565 570 575 cct ggg cag cag atg acc ctg aag ata gag ggt gac cac ggg gcc cgg 1836 Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg 580 585 590 gtg gta ctg gtg gcc gtg gac aag ggc gtg ttc gtg ctg aat aag aag 1884 Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys 595 600 605 aac aaa ctg acg cag agt aag atc tgg gac gtg gtg gag aag gca gac 1932 Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp 610 615 620 atc ggc tgc acc ccg ggc agt ggg aag gat tac gcc ggt gtc ttc tcc 1980 Ile Gly Cys Thr Pro Gly Ser Gly Lys Asp Tyr Ala Gly Val Phe Ser 625 630 635 640 gac gca ggg ctg acc ttc acg agc agc agt ggc cag cag acc gcc cag 2028 Asp Ala Gly Leu Thr Phe Thr Ser Ser Ser Gly Gln Gln Thr Ala Gln 645 650 655 agg gca gaa ctt cag tgc ccg cag cca gcc gcc cgc cga cgc cgt tcc 2076 Arg Ala Glu Leu Gln Cys Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser 660 665 670 gtg cag ctc acg gag aag cga atg gac aaa gtc ggc aag tac ccc aag 2124 Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 675 680 685 gag ctg cgc aag tgc tgc gag gac ggc atg cgg gag aac ccc atg agg 2172 Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg 690 695 700 ttc tcg tgc cag cgc cgg acc cgt ttc atc tcc ctg ggc gag gcg tgc 2220 Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys 705 710 715 720 aag aag gtc ttc ctg gac tgc tgc aac tac atc aca gag ctg cgg cgg 2268 Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg Arg 725 730 735 cag cac gcg cgg gcc agc cac ctg ggc ctg gcc agg agt aac ctg gat 2316 Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg Ser Asn Leu Asp 740 745 750 gag gac atc att gca gaa gag aac atc gtt tcc cga agt gag ttc cca 2364 Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser Glu Phe Pro 755 760 765 gag agc tgg ctg tgg aac gtt gag gac ttg aaa gag cca ccg aaa aat 2412 Glu Ser Trp Leu Trp Asn Val Glu Asp Leu Lys Glu Pro Pro Lys Asn 770 775 780 gga atc tct acg aag ctc atg aat ata ttt ttg aaa gac tcc atc acc 2460 Gly Ile Ser Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile Thr 785 790 795 800 acg tgg gag att ctg gct gtc agc atg tcg gac aag aaa ggg atc tgt 2508 Thr Trp Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys 805 810 815 gtg gca gac ccc ttc gag gtc aca gta atg cag gac ttc ttc atc gac 2556 Val Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe Ile Asp 820 825 830 ctg cgg cta ccc tac tct gtt gtt cga aac gag cag gtg gaa atc cga 2604 Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg 835 840 845 gcc gtt ctc tac aat tac cgg cag aac caa gag ctc aag gtg agg gtg 2652 Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val Arg Val 850 855 860 gaa cta ctc cac aat cca gcc ttc tgc agc ctg gcc acc acc aag agg 2700 Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala Thr Thr Lys Arg 865 870 875 880 cgt cac cag cag acc gta acc atc ccc ccc aag tcc tcg ttg tcc gtt 2748 Arg His Gln Gln Thr Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val 885 890 895 cca tat gtc atc gtg ccg cta aag acc ggc ctg cag gaa gtg gaa gtc 2796 Pro Tyr Val Ile Val Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val 900 905 910 aag gct gcc gtc tac cat cat ttc atc agt gac ggt gtc agg aag tcc 2844 Lys Ala Ala Val Tyr His His Phe Ile Ser Asp Gly Val Arg Lys Ser 915 920 925 ctg aag gtc gtg ccg gaa gga atc aga atg aac aaa act gtg gct gtt 2892 Leu Lys Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val 930 935 940 cgc acc ctg gat cca gaa cgc ctg ggc cgt gaa gga gtg cag aaa gag 2940 Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln Lys Glu 945 950 955 960 gac atc cca cct gca gac ctc agt gac caa gtc ccg gac acc gag tct 2988 Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu Ser 965 970 975 gag acc aga att ctc ctg caa ggg acc cca gtg gcc cag atg aca gag 3036 Glu Thr Arg Ile Leu Leu Gln Gly Thr Pro Val Ala Gln Met Thr Glu 980 985 990 gat gcc gtc gac gcg gaa cgg ctg aag cac ctc att gtg acc ccc tcg 3084 Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu Ile Val Thr Pro Ser 995 1000 1005 ggc tgc ggg gaa cag aac atg atc ggc atg acg ccc acg gtc atc gct 3132 Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile Ala 1010 1015 1020 gtg cat tac ctg gat gaa acg gag cag tgg gag aag ttc ggc cta gag 3180 Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe Gly Leu Glu 1025 1030 1035 1040 aag cgg cag ggg gcc ttg gag ctc atc aag aag ggg tac acc cag cag 3228 Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln 1045 1050 1055 ctg gcc ttc aga caa ccc agc tct gcc ttt gcg gcc ttc gtg aaa cgg 3276 Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala Phe Val Lys Arg 1060 1065 1070 gca ccc agc acc tgg ctg acc gcc tac gtg gtc aag gtc ttc tct ctg 3324 Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val Phe Ser Leu 1075 1080 1085 gct gtc aac ctc atc gcc atc gac tcc caa gtc ctc tgc ggg gct gtt 3372 Ala Val Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys Gly Ala Val 1090 1095 1100 aaa tgg ctg atc ctg gag aag cag aag ccc gac ggg gtc ttc cag gag 3420 Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Gln Glu 1105 1110 1115 1120 gat gcg ccc gtg ata cac caa gaa atg att ggt gga tta cgg aac aac 3468 Asp Ala Pro Val Ile His Gln Glu Met Ile Gly Gly Leu Arg Asn Asn 1125 1130 1135 aac gag aaa gac atg gcc ctc acg gcc ttt gtt ctc atc tcg ctg cag 3516 Asn Glu Lys Asp Met Ala Leu Thr Ala Phe Val Leu Ile Ser Leu Gln 1140 1145 1150 gag gct aaa gat att tgc gag gag cag gtc aac agc ctg cca ggc agc 3564 Glu Ala Lys Asp Ile Cys Glu Glu Gln Val Asn Ser Leu Pro Gly Ser 1155 1160 1165 atc act aaa gca gga gac ttc ctt gaa gcc aac tac atg aac cta cag 3612 Ile Thr Lys Ala Gly Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln 1170 1175 1180 aga tcc tac act gtg gcc att gct ggc tat gct ctg gcc cag atg ggc 3660 Arg Ser Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly 1185 1190 1195 1200 agg ctg aag ggg cct ctt ctt aac aaa ttt ctg acc aca gcc aaa gat 3708 Arg Leu Lys Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp 1205 1210 1215 aag aac cgc tgg gag gac cct ggt aag cag ctc tac aac gtg gag gcc 3756 Lys Asn Arg Trp Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val Glu Ala 1220 1225 1230 aca tcc tat gcc ctc ttg gcc cta ctg cag cta aaa gac ttt gac ttt 3804 Thr Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp Phe Asp Phe 1235 1240 1245 gtg cct ccc gtc gtg cgt tgg ctc aat gaa cag aga tac tac ggt ggt 3852 Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly 1250 1255 1260 ggc tat ggc tct acc cag gcc acc ttc atg gtg ttc caa gcc ttg gct 3900 Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala 1265 1270 1275 1280 caa tac caa aag gac gcc cct gac cac cag gaa ctg aac ctt gat gtg 3948 Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn Leu Asp Val 1285 1290 1295 tcc ctc caa ctg ccc agc cgc agc tcc aag atc acc cac cgt atc cac 3996 Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys Ile Thr His Arg Ile His 1300 1305 1310 tgg gaa tct gcc agc ctc ctg cga tca gaa gag acc aag gaa aat gag 4044 Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu Thr Lys Glu Asn Glu 1315 1320 1325 ggt ttc aca gtc aca gct gaa gga aaa ggc caa ggc acc ttg tcg gtg 4092 Gly Phe Thr Val Thr Ala Glu Gly Lys Gly Gln Gly Thr Leu Ser Val 1330 1335 1340 gtg aca atg tac cat gct aag gcc aaa gat caa ctc acc tgt aat aaa 4140 Val Thr Met Tyr His Ala Lys Ala Lys Asp Gln Leu Thr Cys Asn Lys 1345 1350 1355 1360 ttc gac ctc aag gtc acc ata aaa cca gca ccg gaa aca gaa aag agg 4188 Phe Asp Leu Lys Val Thr Ile Lys Pro Ala Pro Glu Thr Glu Lys Arg 1365 1370 1375 cct cag gat gcc aag aac act atg atc ctt gag atc tgt acc agg tac 4236 Pro Gln Asp Ala Lys Asn Thr Met Ile Leu Glu Ile Cys Thr Arg Tyr 1380 1385 1390 cgg gga gac cag gat gcc act atg tct ata ttg gac ata tcc atg atg 4284 Arg Gly Asp Gln Asp Ala Thr Met Ser Ile Leu Asp Ile Ser Met Met 1395 1400 1405 act ggc ttt gct cca gac aca gat gac ctg aag cag ctg gcc aat ggt 4332 Thr Gly Phe Ala Pro Asp Thr Asp Asp Leu Lys Gln Leu Ala Asn Gly 1410 1415 1420 gtt gac aga tac atc tcc aag tat gag ctg gac aaa gcc ttc tcc gat 4380 Val Asp Arg Tyr Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp 1425 1430 1435 1440 agg aac acc ctc atc atc tac ctg gac aag gtc tca cac tct gag gat 4428 Arg Asn Thr Leu Ile Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp 1445 1450 1455 gac tgt cta gct ttc aaa gtt cac caa tac ttt aat gta gag ctt atc 4476 Asp Cys Leu Ala Phe Lys Val His Gln Tyr Phe Asn Val Glu Leu Ile 1460 1465 1470 cag cct gga gca gtc aag gtc tac gcc tat tac aac ctg gag gaa agc 4524 Gln Pro Gly Ala Val Lys Val Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser 1475 1480 1485 tgt acc cgg ttc tac cat ccg gaa aag gag gat gga aag ctg aac aag 4572 Cys Thr Arg Phe Tyr His Pro Glu Lys Glu Asp Gly Lys Leu Asn Lys 1490 1495 1500 ctc tgc cgt gat gaa ctg tgc cgc tgt gct gag gag aat tgc ttc ata 4620 Leu Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys Phe Ile 1505 1510 1515 1520 caa aag tcg gat gac aag gtc acc ctg gaa gaa cgg ctg gac aag gcc 4668 Gln Lys Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu Asp Lys Ala 1525 1530 1535 tgt gag cca gga gtg gac tat gtg tac aag acc cga ctg gtc aag gtt 4716 Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Arg Leu Val Lys Val 1540 1545 1550 cag ctg tcc aat gac ttt gac gag tac atc atg gcc att gag cag acc 4764 Gln Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met Ala Ile Glu Gln Thr 1555 1560 1565 atc aag tca ggc tcg gat gag gtg cag gtt gga cag cag cgc acg ttc 4812 Ile Lys Ser Gly Ser Asp Glu Val Gln Val Gly Gln Gln Arg Thr Phe 1570 1575 1580 atc agc ccc atc aag tgc aga gaa gcc ctg aag ctg gag gag aag aaa 4860 Ile Ser Pro Ile Lys Cys Arg Glu Ala Leu Lys Leu Glu Glu Lys Lys 1585 1590 1595 1600 cac tac ctc atg tgg ggt ctc tcc tcc gat ttc tgg gga gag aag ccc 4908 His Tyr Leu Met Trp Gly Leu Ser Ser Asp Phe Trp Gly Glu Lys Pro 1605 1610 1615 aac ctc agc tac atc atc ggg aag gac act tgg gtg gag cac tgg cct 4956 Asn Leu Ser Tyr Ile Ile Gly Lys Asp Thr Trp Val Glu His Trp Pro 1620 1625 1630 gag gag gac gaa tgc caa gac gaa gag aac cag aaa caa tgc cag gac 5004 Glu Glu Asp Glu Cys Gln Asp Glu Glu Asn Gln Lys Gln Cys Gln Asp 1635 1640 1645 ctc ggc gcc ttc acc gag agc atg gtt gtc ttt ggg tgc ccc aac tga 5052 Leu Gly Ala Phe Thr Glu Ser Met Val Val Phe Gly Cys Pro Asn 1650 1655 1660 ccacaccccc attcc 5067 <210> SEQ ID NO 4 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 4 cgtgatacac caagaaatga ttgg 24 <210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 ctgcagcgag atgagaacaa ag 22 <210> SEQ ID NO 6 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 6 acaacgagaa agacatggcc ctcacg 26 <210> SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 7 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaagatggtg atgggatttc 20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 9 caagcttccc gttctcagcc 20 <210> SEQ ID NO 10 <211> LENGTH: 6435 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <400> SEQUENCE: 10 aagcttagga aactatgttg cgaaattttg ggcagtccct ggtgcaggaa cagggaggga 60 ccagagagga gagccatata aagagccagc ggctacagcc ccagctcgcc tctgcccacc 120 cctgcccctt accccttcat tccttccacc tttttccttc actatgggac cagcttcagg 180 gtcccagcta ctagtgctac tgctgctgtt ggccagctcc ccattagctc tggggatccc 240 catgtaagta gttacctttt gggggtgcag tttcttatac aatttaggag tcacctaggt 300 gagtcaccta ggagtcaccc acttgggggg agacagggat gttaagaatt tgtgctgggg 360 gctggaggat ggctcagtgg gtatgaagtc ttgctgcatg gacataagga ccttaactca 420 aactcccagc acccacagaa aagccaggag tggcctccag agcctgtaac cccacactgt 480 ggggctggag accagatact ggggcttgct gactgccagc ccagtgccag gttcagagag 540 agatgttgac tcaaggtgtg gggagggagg ggcatagaac aggacaccgg acatcttcct 600 gtgacgtgac atacatacat acatacatac atacatacat acatacatac aaacagagag 660 aaagagagag aatgtgagtg tttgggttgt cttatgttca tagaactcag gtattgtagc 720 attggtgtgc ctacttatga agaagaaggt ctatagattt atggtgtctg tgatgtcttg 780 tggtttaggc aatggtaagt tttaagatgt gaggactgga gggttgttgg ggtctatagc 840 ccttgtgtgg actgtcaaaa gccaagctca gcattgggcc tgattacatg gacctgtcat 900 cctgtccact ctggaagccc aggcaggaag tgtacacatt ccagccaagc ctgggctaca 960 gagtgagttc agggctagcc tgaacaactt agtgagatgc tgtttccaaa cagaaaagtg 1020 aggagaggct gaggtatagc ttagtggtag agagagcact ttctcagtac aagcaaagtc 1080 cttatggctg gtacccagtg ctgaagccag gaaaagacag cttctagtgg ctaagggaaa 1140 gtgggctgtg gtttggtacc ttcttgtgtg gggaggacgt ggatccaggc atctgctcca 1200 gcactgagaa ctagaacacg ctagaactag ggactgggcc agccagagtg agggctgaag 1260 tctcaggcct tgaggtgaca tctgctccca caccccacgg tcaggtattc catcattact 1320 cccaatgtcc tacggctgga gagcgaagag accatcgtac tggaggccca cgatgctcag 1380 ggtgacatcc cagtcacagt cactgtgcaa gacttcctaa agaggcaagt gctgaccagt 1440 gagaagacag tgttgacagg agccagtgga catctgagaa gcgtctccat caaggtgggc 1500 aaggaactgg aactacagtc agccgtagcc ccttccggtc cggcctgggt ccctcaggct 1560 gcctcctata gcctcctcgg agctcctccc ttctgagtcc ctcccctctg acgcccctgc 1620 cgtctgggtc cctctcgttt gatctcctcc cctctgagtc ccctaccctt tgagaccctc 1680 tgctctgagc cccttccctc tgagattcct cctctcagtc cctctcctct tgagtctctt 1740 cctccttgag cccctcctgt ctgaggggag atgacagaga ggaggcccag ggggatctag 1800 gggatgcttt ctgggcacca ctccctgaca cagactcctg acatcccacg catagattcc 1860 agccagtaag gaattcaact cagataagga ggggcacaag tacgtgacag tggtggcaaa 1920 cttcggggaa acggtggtgg agaaagcagt gatggtaagc ttccagagtg ggtacctctt 1980 catccagaca gacaagacca tctacacccc tggctccact gtcttatatc ggatcttcac 2040 tgtggacaac aacctactgc ccgtgggcaa gacagtcgtc atcctcattg agacccccga 2100 tggcattcct gtcaagagag acattctgtc ttccaacaac caacacggca tcttgccttt 2160 gtcttggaac attcctgaac tggtcaacat ggggcagtgg aagatccgag ccttttacga 2220 acatgcgccg aagcagatct tctccgcaga gtttgaggtg aaggaatacg tgctgcccag 2280 ttttgaggtc cgggtggagc ccacagagac attttattac atcgatgacc caaatggcct 2340 ggaagtttcc atcatagcca agttcctgta cgggaaaaac gtggacggga cagccttcgt 2400 gatttttggg gtccaggatg gcgataagaa gatttctctg gcccactccc tcacgcgcgt 2460 agtgattgag gatggtgtgg gggatgcagt gctgacccgg aaggtgctga tggagggggt 2520 acggccttcc aacgccgacg ccctggtggg gaagtccctg tatgtctccg tcactgtcat 2580 cctgcactca ggtagtgaca tggtagaggc agagcgcagt gggatcccga ttgtcacttc 2640 cccgtaccag atccacttca ccaagacacc caaattcttc aagccagcca tgccctttga 2700 cctcatggtg ttcgtgacca accccgatgg ctctccggcc agcaaagtgc tggtggtcac 2760 tcagggatct aatgcaaagg ctctcaccca agatgatggc gtggccaagc taagcatcaa 2820 cacacccaac agccgccaac ccctgaccat cacagtccgc accaagaagg acactctccc 2880 agaatcacgg caggccacca agacaatgga ggcccatccc tacagcacta tgcacaactc 2940 caacaactac ctacacttgt cagtgtcacg aatggagctc aagccggggg acaacctcaa 3000 tgtcaacttc cacctgcgca cagacccagg ccatgaggcc aagatccgat actacaccta 3060 cctggttatg aacaagggga agctcctgaa ggcaggccgc caggttcggg agcctggcca 3120 ggacctggtg gtcttgtccc tgcccatcac tccagagttt attccttcat ttcgcctggt 3180 ggcttactac accctgattg gagctagtgg ccagagggag gtggtggctg actctgtgtg 3240 ggtggatgtg aaggattcct gtattggcac gctggtggtg aagggtgacc caagagataa 3300 ccatctcgca cctgggcaac aaacgacact caggattgaa ggaaaccagg gggcccgagt 3360 ggggctagtg gctgtggaca agggagtgtt tgtgctgaac aagaagaaca aactcacaca 3420 gagcaagatc tgggatgtgg tagagaaggc agacattggc tgcaccccag gcagtgggaa 3480 gaactatgct ggtgtcttca tggatgcagg cctggccttc aagacaagcc aaggactgca 3540 gactgaacag agagcagatc ttgagtgcac caagccagca gcccgccgcc gtcgctcagt 3600 acagttgatg gaaagaagga tggacaaagc tggtcagtac actgacaagg gtcttcggaa 3660 gtgttgtgag gatggtatgc gggatatccc tatgagatac agctgccagc gccgggcacg 3720 cctcatcacc cagggcgaga actgcataaa ggccttcata gactgctgca accacatcac 3780 caagctgcgt gaacaacaca gaagagacca cgtgctgggc ctggccagga gtgaattgga 3840 ggaagacata attccagaag aagatattat ctctagaagc cacttcccac agagctggtt 3900 gtggaccata gaagagttga aagaaccaga gaaaaatgga atctctacga aggtcatgaa 3960 catctttctc aaagattcca tcaccacctg ggagattctg gcagtgagct tgtcagacaa 4020 gaaagggatc tgtgtggcag acccctatga gatcagagtg atgcaggact tcttcattga 4080 cctgcggctg ccctactctg tagtgcgcaa cgaacaggtg gagatcagag ctgtgctctt 4140 caactaccgt gaacaggagg aacttaaggt gagggtggaa ctgttgcata atccagcctt 4200 ctgcagcatg gccaccgcca agaatcgcta cttccagacc atcaaaatcc ctcccaagtc 4260 ctcggtggct gtaccgtatg tcattgtccc cttgaagatc ggccaacaag aggtggaggt 4320 caaggctgct gtcttcaatc acttcatcag tgatggtgtc aagaagacac tgaaggtcgt 4380 gccagaagga atgagaatca acaaaactgt ggccatccat acactggacc cagagaagct 4440 cggtcaaggg ggagtgcaga aggtggatgt gcctgccgca gaccttagcg accaagtgcc 4500 agacacagac tctgagacca gaattatcct gcaagggagc ccggtggttc agatggctga 4560 agatgctgtg gacggggagc ggctgaaaca cctgatcgtg acccccgcag gctgtgggga 4620 acagaacatg attggcatga caccaacagt cattgcggta cactacctgg accagaccga 4680 acagtgggag aagttcggca tagagaagag gcaagaggcc ctggagctca tcaagaaagg 4740 gtacacccag cagctggcct tcaaacagcc cagctctgcc tatgctgcct tcaacaaccg 4800 gccccccagc acctggctga cagcctacgt ggtcaaggtc ttctctctag ctgccaacct 4860 catcgccatc gactctcacg tcctgtgtgg ggctgttaaa tggttgattc tggagaaaca 4920 gaagccggat ggtgtctttc aggaggatgg gcccgtgatt caccaagaaa tgattggtgg 4980 cttccggaac gccaaggagg cagatgtgtc actcacagcc ttcgtcctca tcgcactgca 5040 ggaagccagg gacatctgtg aggggcaggt caatagcctt cctgggagca tcaacaaggc 5100 aggggagtat attgaagcca gttacatgaa cctgcagaga ccatacacag tggccattgc 5160 tgggtatgcc ctggccctga tgaacaaact ggaggaacct tacctcggca agtttctgaa 5220 cacagccaaa gatcggaacc gctgggagga gcctgaccag cagctctaca acgtagaggc 5280 cacatcctac gccctcctgg ccctgctgct gctgaaagac tttgactctg tgccccctgt 5340 agtgcgctgg ctcaatgagc aaagatacta cggaggcggc tatggctcca cccaggctac 5400 cttcatggta ttccaagcct tggcccaata tcaaacagat gtccctgacc ataaggactt 5460 gaacatggat gtgtccttcc acctccccag ccgtagctct gcaaccacgt ttcgcctgct 5520 ctgggaaaat ggcaacctcc tgcgatcgga agagaccaag caaaatgagg ccttctctct 5580 aacagccaaa ggaaaaggcc gaggcacatt gtcggtggtg gcagtgtatc atgccaaact 5640 caaaagcaaa gtcacctgca agaagtttga cctcagggtc agcataagac cagcccctga 5700 gacagccaag aagcccgagg aagccaagaa taccatgttc cttgaaatct gcaccaagta 5760 cttgggagat gtggacgcca ctatgtccat cctggacatc tccatgatga ctggctttgc 5820 tccagacaca aaggacctgg aactgctggc ctctggagta gatagataca tctccaagta 5880 cgagatgaac aaagccttct ccaacaagaa caccctcatc atctacctag aaaagatttc 5940 acacaccgaa gaagactgcc tgaccttcaa agttcaccag tactttaatg tgggacttat 6000 ccagcccggg tcggtcaagg tctactccta ttacaacctc gaggaatcat gcacccggtt 6060 ctatcatcca gagaaggacg atgggatgct cagcaagctg tgccacagtg aaatgtgccg 6120 gtgtgctgaa gagaactgct tcatgcaaca gtcacaggag aagatcaacc tgaatgtccg 6180 gctagacaag gcttgtgagc ccggagtcga ctatgtgtac aagaccgagc taaccaacat 6240 aaagctgttg gatgattttg atgagtacac catgaccatc cagcaggtca tcaagtcagg 6300 ctcagatgag gtgcaggcag ggcagcaacg caagttcatc agccacatca agtgcagaaa 6360 cgccctgaag ctgcagaaag ggaagaagta cctcatgtgg ggcctctcct ctgacctctg 6420 gggagaaaag cccaa 6435 <210> SEQ ID NO 11 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 11 aagctgtgcc acagtgaaat gt 22 <210> SEQ ID NO 12 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 12 cgactccggg ctcacaag 18 <210> SEQ ID NO 13 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 13 tagccggaca ttcaggttga tcttctcct 29 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 14 ggcaaattca acggcacagt 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 15 gggtctcgct cctggaagat 20 <210> SEQ ID NO 16 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 16 aaggccgaga atgggaagct tgtcatc 27 <210> SEQ ID NO 17 <211> LENGTH: 607 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <400> SEQUENCE: 17 tttctcccat tctacttccc tcctcagcat tggaagctcg taagtgggct ctgactccca 60 gcctacagag agattcctag gaagtggttc gactgataaa cgcatggcca aaagtgaact 120 ggggatgagg tccaagacat ctgcggtggg gggttctcca gaccttagtg ttcttccact 180 acaaagtggg tccaacagag aaaggtctgt gttcaccagg tggccctgac cctgggagag 240 tccagggcag ggtgcagctg cattcatgct gctggggaac atgccctcag gttactcacc 300 ccatggacat gttggcccca gggactgaaa agcttaggaa atggtattga gaaatctggg 360 gcagccaaaa ggggagaggc catggggaga agggggggct gagtggggga aagcaggagc 420 cagataaaaa gccagctcca gcaggcgctg ctcactcctc cccatcctct ccctctgtcc 480 ctctgtccct ctgaccctgc actgtcccag caccatggga cccacctcag gtcccagcct 540 gctgctcctg ctactaaccc acctccccct ggctctgggg agtcccatgt gagtggttat 600 gactcta 607 <210> SEQ ID NO 18 <220> FEATURE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 18 000 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 19 ccatggtgct gggacagtgc 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 20 aggtgggtcc catggtgctg 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 21 ttagtagcag gagcagcagg 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 22 ctgttggacc cactttgtag 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 23 gtcgtgggcc tccagcacca 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 gtaacagtga ctggaacatc 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 ctgccctgga ctctcccagg 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 actggacagc actagttttt 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 cccagcagca tgaatgcagc 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 ctgagggcat gttccccagc 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 tgaactccct gttggctggg 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 tctgtctgga tgaagaggta 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 tcttgtctgt ctggatgaag 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 gatggtcttg tctgtctgga 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 gtgtagatgg tcttgtctgt 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 ttgtggttga cggtgaagat 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 cacgtactcc ttcacctcaa 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 36 aatttctctg taggctccac 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 37 agaggaacct ggcggtgatg 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 38 tcgccatcct ggatcccgaa 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 39 tcaatcggaa tgcgcttgag 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 40 gacttcccca ccaggtcttc 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 41 tgcctgcacc atgtcactgc 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 42 ttggtgaagt ggatctggta 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 43 ggtgtcttgg tgaagtggat 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 caccatgagg tcaaagggca 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 tcacgaacac catgaggtca 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 ggctggagag ccatcagggt 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 ctgcctccga gagctcctgc 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 ttgttggagt tgcccacggt 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 tgtacgtagc actgagagat 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 caggtcctgg ccgggctctc 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 cagcgtgtag tacgccacca 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 tattcagcac gaacacgccc 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 ccgcagctct gtgatgtagt 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 caggcccagg tggctggccc 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 tcgtagagat tccatttttc 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 cccacgtggt gatggagtct 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 ccctttcttg tccgacatgc 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 cacacagatc cctttcttgt 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 ggtctgccac acagatccct 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 ttcctgcagg ccggtcttta 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 acggcagcct tgacttccac 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 acttggtcac tgaggtctgc 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 caggagaatt ctggtctcag 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 ccttgcagga gaattctggt 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 gggtcccttg caggagaatt 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 tccaggtaat gcacagcgat 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 gccagctgct gggtgtaccc 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 tgaaggccag ctgctgggtg 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 aagaccttga ccacgtaggc 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 agagagaaga ccttgaccac 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 agtcgatggc gatgaggttg 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 72 gggcttctgc ttctccagga 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 gaagaccccg tcgggcttct 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 ttcttggtgt atcacgggcg 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 ccaccaatca tttcttggtg 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 tcgcaaatat ctttagcctc 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 gctgttgacc tgctcctcgc 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 78 cagtgtagga tctctgtagg 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 79 cttatctttg gctgtggtca 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 80 taccagggtc ctcccagcgg 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 81 gcataggatg tggcctccac 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 82 tggaacacca tgaaggtggc 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 83 gtattgagcc aaggcttgga 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 84 ggtgatcttg gagctgcggc 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 85 gttcttggca tcctgaggcc 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 86 gcaaagccag tcatcatgga 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 87 ctggagcaaa gccagtcatc 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 88 tgtgtctgga gcaaagccag 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 89 gaaggctttg tccagctcat 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 90 ggtagatgat gagggtgttc 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 91 ctcagagtgt gagaccttgt 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 92 ccttttccgg atggtagaac 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 93 gtactcgtca aagtcattgg 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 94 ccacatgagg tagtgtttct 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 95 tcggaggaga gaccccacat 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 96 tcaggccagt gctccaccca 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 97 ggcatcacac acgaagccca 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 98 tgcggcagcc gctgcccatg 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 99 gtcatagctg tggcagcctg 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 100 ggttgagctc cacatctgcc 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 101 ttgaagaaat cctccttgtc 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 102 atgcattcga tgcggcagcc 20 <210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 103 caaacagctc tcgggagatg 20 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 104 tagctcatgc ctgtcttgca 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 105 ggagggacta cccacagcaa 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 106 actcgctgaa gaatatgcat 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 107 catcatactc ctgggccacc 20 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 108 aacttctgct tgctctcctc 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 109 tgggcaccaa cttctgcttg 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 110 tctgcttgct ctcctccatg 20 <210> SEQ ID NO 111 <211> LENGTH: 5087 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (57)...(5048) <400> SEQUENCE: 111 gcctctgccc acccctgccc cttacccctt cattccttcc acctttttcc ttcact atg 59 Met 1 gga cca gct tca ggg tcc cag cta cta gtg cta ctg ctg ctg ttg gcc 107 Gly Pro Ala Ser Gly Ser Gln Leu Leu Val Leu Leu Leu Leu Leu Ala 5 10 15 agc tcc cca tta gct ctg ggg atc ccc atg tat tcc atc att act ccc 155 Ser Ser Pro Leu Ala Leu Gly Ile Pro Met Tyr Ser Ile Ile Thr Pro 20 25 30 aat gtc cta cgg ctg gag agc gaa gag acc atc gta ctg gag gcc cac 203 Asn Val Leu Arg Leu Glu Ser Glu Glu Thr Ile Val Leu Glu Ala His 35 40 45 gat gct cag ggt gac atc cca gtc aca gtc act gtg caa gac ttc cta 251 Asp Ala Gln Gly Asp Ile Pro Val Thr Val Thr Val Gln Asp Phe Leu 50 55 60 65 aag agg caa gtg ctg acc agt gag aag aca gtg ttg aca gga gcc agt 299 Lys Arg Gln Val Leu Thr Ser Glu Lys Thr Val Leu Thr Gly Ala Ser 70 75 80 gga cat ctg aga agc gtc tcc atc aag att cca gcc agt aag gaa ttc 347 Gly His Leu Arg Ser Val Ser Ile Lys Ile Pro Ala Ser Lys Glu Phe 85 90 95 aac tca gat aag gag ggg cac aag tac gtg aca gtg gtg gca aac ttc 395 Asn Ser Asp Lys Glu Gly His Lys Tyr Val Thr Val Val Ala Asn Phe 100 105 110 ggg gaa acg gtg gtg gag aaa gca gtg atg gta agc ttc cag agt ggg 443 Gly Glu Thr Val Val Glu Lys Ala Val Met Val Ser Phe Gln Ser Gly 115 120 125 tac ctc ttc atc cag aca gac aag acc atc tac acc cct ggc tcc act 491 Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr 130 135 140 145 gtc tta tat cgg atc ttc act gtg gac aac aac cta ctg ccc gtg ggc 539 Val Leu Tyr Arg Ile Phe Thr Val Asp Asn Asn Leu Leu Pro Val Gly 150 155 160 aag aca gtc gtc atc ctc att gag acc ccc gat ggc att cct gtc aag 587 Lys Thr Val Val Ile Leu Ile Glu Thr Pro Asp Gly Ile Pro Val Lys 165 170 175 aga gac att ctg tct tcc aac aac caa cac ggc atc ttg cct ttg tct 635 Arg Asp Ile Leu Ser Ser Asn Asn Gln His Gly Ile Leu Pro Leu Ser 180 185 190 tgg aac att cct gaa ctg gtc aac atg ggg cag tgg aag atc cga gcc 683 Trp Asn Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 195 200 205 ttt tac gaa cat gcg ccg aag cag atc ttc tcc gca gag ttt gag gtg 731 Phe Tyr Glu His Ala Pro Lys Gln Ile Phe Ser Ala Glu Phe Glu Val 210 215 220 225 aag gaa tac gtg ctg ccc agt ttt gag gtc cgg gtg gag ccc aca gag 779 Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Arg Val Glu Pro Thr Glu 230 235 240 aca ttt tat tac atc gat gac cca aat ggc ctg gaa gtt tcc atc ata 827 Thr Phe Tyr Tyr Ile Asp Asp Pro Asn Gly Leu Glu Val Ser Ile Ile 245 250 255 gcc aag ttc ctg tac ggg aaa aac gtg gac ggg aca gcc ttc gtg att 875 Ala Lys Phe Leu Tyr Gly Lys Asn Val Asp Gly Thr Ala Phe Val Ile 260 265 270 ttt ggg gtc cag gat ggc gat aag aag att tct ctg gcc cac tcc ctc 923 Phe Gly Val Gln Asp Gly Asp Lys Lys Ile Ser Leu Ala His Ser Leu 275 280 285 acg cgc gta gtg att gag gat ggt gtg ggg gat gca gtg ctg acc cgg 971 Thr Arg Val Val Ile Glu Asp Gly Val Gly Asp Ala Val Leu Thr Arg 290 295 300 305 aag gtg ctg atg gag ggg gta cgg cct tcc aac gcc gac gcc ctg gtg 1019 Lys Val Leu Met Glu Gly Val Arg Pro Ser Asn Ala Asp Ala Leu Val 310 315 320 ggg aag tcc ctg tat gtc tcc gtc act gtc atc ctg cac tca ggt agt 1067 Gly Lys Ser Leu Tyr Val Ser Val Thr Val Ile Leu His Ser Gly Ser 325 330 335 gac atg gta gag gca gag cgc agt ggg atc ccg att gtc act tcc ccg 1115 Asp Met Val Glu Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 340 345 350 tac cag atc cac ttc acc aag aca ccc aaa ttc ttc aag cca gcc atg 1163 Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Phe Phe Lys Pro Ala Met 355 360 365 ccc ttt gac ctc atg gtg ttc gtg acc aac ccc gat ggc tct ccg gcc 1211 Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 370 375 380 385 agc aaa gtg ctg gtg gtc act cag gga tct aat gca aag gct ctc acc 1259 Ser Lys Val Leu Val Val Thr Gln Gly Ser Asn Ala Lys Ala Leu Thr 390 395 400 caa gat gat ggc gtg gcc aag cta agc atc aac aca ccc aac agc cgc 1307 Gln Asp Asp Gly Val Ala Lys Leu Ser Ile Asn Thr Pro Asn Ser Arg 405 410 415 caa ccc ctg acc atc aca gtc cgc acc aag aag gac act ctc cca gaa 1355 Gln Pro Leu Thr Ile Thr Val Arg Thr Lys Lys Asp Thr Leu Pro Glu 420 425 430 tca cgg cag gcc acc aag aca atg gag gcc cat ccc tac agc act atg 1403 Ser Arg Gln Ala Thr Lys Thr Met Glu Ala His Pro Tyr Ser Thr Met 435 440 445 cac aac tcc aac aac tac cta cac ttg tca gtg tca cga atg gag ctc 1451 His Asn Ser Asn Asn Tyr Leu His Leu Ser Val Ser Arg Met Glu Leu 450 455 460 465 aag ccg ggg gac aac ctc aat gtc aac ttc cac ctg cgc aca gac cca 1499 Lys Pro Gly Asp Asn Leu Asn Val Asn Phe His Leu Arg Thr Asp Pro 470 475 480 ggc cat gag gcc aag atc cga tac tac acc tac ctg gtt atg aac aag 1547 Gly His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Val Met Asn Lys 485 490 495 ggg aag ctc ctg aag gca ggc cgc cag gtt cgg gag cct ggc cag gac 1595 Gly Lys Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln Asp 500 505 510 ctg gtg gtc ttg tcc ctg ccc atc act cca gag ttt att cct tca ttt 1643 Leu Val Val Leu Ser Leu Pro Ile Thr Pro Glu Phe Ile Pro Ser Phe 515 520 525 cgc ctg gtg gct tac tac acc ctg att gga gct agt ggc cag agg gag 1691 Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg Glu 530 535 540 545 gtg gtg gct gac tct gtg tgg gtg gat gtg aag gat tcc tgt att ggc 1739 Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Ile Gly 550 555 560 acg ctg gtg gtg aag ggt gac cca aga gat aac cat ctc gca cct ggg 1787 Thr Leu Val Val Lys Gly Asp Pro Arg Asp Asn His Leu Ala Pro Gly 565 570 575 caa caa acg aca ctc agg att gaa gga aac cag ggg gcc cga gtg ggg 1835 Gln Gln Thr Thr Leu Arg Ile Glu Gly Asn Gln Gly Ala Arg Val Gly 580 585 590 cta gtg gct gtg gac aag gga gtg ttt gtg ctg aac aag aag aac aaa 1883 Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys Asn Lys 595 600 605 ctc aca cag agc aag atc tgg gat gtg gta gag aag gca gac att ggc 1931 Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp Ile Gly 610 615 620 625 tgc acc cca ggc agt ggg aag aac tat gct ggt gtc ttc atg gat gca 1979 Cys Thr Pro Gly Ser Gly Lys Asn Tyr Ala Gly Val Phe Met Asp Ala 630 635 640 ggc ctg gcc ttc aag aca agc caa gga ctg cag act gaa cag aga gca 2027 Gly Leu Ala Phe Lys Thr Ser Gln Gly Leu Gln Thr Glu Gln Arg Ala 645 650 655 gat ctt gag tgc acc aag cca gca gcc cgc cgc cgt cgc tca gta cag 2075 Asp Leu Glu Cys Thr Lys Pro Ala Ala Arg Arg Arg Arg Ser Val Gln 660 665 670 ttg atg gaa aga agg atg gac aaa gct ggt cag tac act gac aag ggt 2123 Leu Met Glu Arg Arg Met Asp Lys Ala Gly Gln Tyr Thr Asp Lys Gly 675 680 685 ctt cgg aag tgt tgt gag gat ggt atg cgg gat atc cct atg aga tac 2171 Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Asp Ile Pro Met Arg Tyr 690 695 700 705 agc tgc cag cgc cgg gca cgc ctc atc acc cag ggc gag aac tgc ata 2219 Ser Cys Gln Arg Arg Ala Arg Leu Ile Thr Gln Gly Glu Asn Cys Ile 710 715 720 aag gcc ttc ata gac tgc tgc aac cac atc acc aag ctg cgt gaa caa 2267 Lys Ala Phe Ile Asp Cys Cys Asn His Ile Thr Lys Leu Arg Glu Gln 725 730 735 cac aga aga gac cac gtg ctg ggc ctg gcc agg agt gaa ttg gag gaa 2315 His Arg Arg Asp His Val Leu Gly Leu Ala Arg Ser Glu Leu Glu Glu 740 745 750 gac ata att cca gaa gaa gat att atc tct aga agc cac ttc cca cag 2363 Asp Ile Ile Pro Glu Glu Asp Ile Ile Ser Arg Ser His Phe Pro Gln 755 760 765 agc tgg ttg tgg acc ata gaa gag ttg aaa gaa cca gag aaa aat gga 2411 Ser Trp Leu Trp Thr Ile Glu Glu Leu Lys Glu Pro Glu Lys Asn Gly 770 775 780 785 atc tct acg aag gtc atg aac atc ttt ctc aaa gat tcc atc acc acc 2459 Ile Ser Thr Lys Val Met Asn Ile Phe Leu Lys Asp Ser Ile Thr Thr 790 795 800 tgg gag att ctg gca gtg agc ttg tca gac aag aaa ggg atc tgt gtg 2507 Trp Glu Ile Leu Ala Val Ser Leu Ser Asp Lys Lys Gly Ile Cys Val 805 810 815 gca gac ccc tat gag atc aga gtg atg cag gac ttc ttc att gac ctg 2555 Ala Asp Pro Tyr Glu Ile Arg Val Met Gln Asp Phe Phe Ile Asp Leu 820 825 830 cgg ctg ccc tac tct gta gtg cgc aac gaa cag gtg gag atc aga gct 2603 Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg Ala 835 840 845 gtg ctc ttc aac tac cgt gaa cag cag gaa ctt aag gtg agg gtg gaa 2651 Val Leu Phe Asn Tyr Arg Glu Gln Gln Glu Leu Lys Val Arg Val Glu 850 855 860 865 ctg ttg cat aat cca gcc ttc tgc agc atg gcc acc gcc aag aat cgc 2699 Leu Leu His Asn Pro Ala Phe Cys Ser Met Ala Thr Ala Lys Asn Arg 870 875 880 tac ttc cag acc atc aaa atc cct ccc aag tcc tcg gtg gct gta ccg 2747 Tyr Phe Gln Thr Ile Lys Ile Pro Pro Lys Ser Ser Val Ala Val Pro 885 890 895 tat gtc att gtc ccc ttg aag atc ggc caa caa gag gtg gag gtc aag 2795 Tyr Val Ile Val Pro Leu Lys Ile Gly Gln Gln Glu Val Glu Val Lys 900 905 910 gct gct gtc ttc aat cac ttc atc agt gat ggt gtc aag aag aca ctg 2843 Ala Ala Val Phe Asn His Phe Ile Ser Asp Gly Val Lys Lys Thr Leu 915 920 925 aag gtc gtg cca gaa gga atg aga atc aac aaa act gtg gcc atc cat 2891 Lys Val Val Pro Glu Gly Met Arg Ile Asn Lys Thr Val Ala Ile His 930 935 940 945 aca ctg gac cca gag aag ctc ggt caa ggg gga gtg cag aag gtg gat 2939 Thr Leu Asp Pro Glu Lys Leu Gly Gln Gly Gly Val Gln Lys Val Asp 950 955 960 gtg cct gcc gca gac ctt agc gac caa gtg cca gac aca gac tct gag 2987 Val Pro Ala Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Asp Ser Glu 965 970 975 acc aga att atc ctg caa ggg agc ccg gtg gtt cag atg gct gaa gat 3035 Thr Arg Ile Ile Leu Gln Gly Ser Pro Val Val Gln Met Ala Glu Asp 980 985 990 gct gtg gac ggg gag cgg ctg aaa cac ctg atc gtg acc ccc gca ggc 3083 Ala Val Asp Gly Glu Arg Leu Lys His Leu Ile Val Thr Pro Ala Gly 995 1000 1005 tgt ggg gaa cag aac atg att ggc atg aca cca aca gtc att gcg gta 3131 Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile Ala Val 1010 1015 1020 1025 cac tac ctg gac cag acc gaa cag tgg gag aag ttc ggc ata gag aag 3179 His Tyr Leu Asp Gln Thr Glu Gln Trp Glu Lys Phe Gly Ile Glu Lys 1030 1035 1040 agg caa gag gcc ctg gag ctc atc aag aaa ggg tac acc cag cag ctg 3227 Arg Gln Glu Ala Leu Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln Leu 1045 1050 1055 gcc ttc aaa cag ccc agc tct gcc tat gct gcc ttc aac aac cgg ccc 3275 Ala Phe Lys Gln Pro Ser Ser Ala Tyr Ala Ala Phe Asn Asn Arg Pro 1060 1065 1070 ccc agc acc tgg ctg aca gcc tac gtg gtc aag gtc ttc tct cta gct 3323 Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val Phe Ser Leu Ala 1075 1080 1085 gcc aac ctc atc gcc atc gac tct cac gtc ctg tgt ggg gct gtt aaa 3371 Ala Asn Leu Ile Ala Ile Asp Ser His Val Leu Cys Gly Ala Val Lys 1090 1095 1100 1105 tgg ttg att ctg gag aaa cag aag ccg gat ggt gtc ttt cag gag gat 3419 Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Gln Glu Asp 1110 1115 1120 ggg ccc gtg att cac caa gaa atg att ggt ggc ttc cgg aac gcc aag 3467 Gly Pro Val Ile His Gln Glu Met Ile Gly Gly Phe Arg Asn Ala Lys 1125 1130 1135 gag gca gat gtg tca ctc aca gcc ttc gtc ctc atc gca ctg cag gaa 3515 Glu Ala Asp Val Ser Leu Thr Ala Phe Val Leu Ile Ala Leu Gln Glu 1140 1145 1150 gcc agg gac atc tgt gag ggg cag gtc aat agc ctt cct ggg agc atc 3563 Ala Arg Asp Ile Cys Glu Gly Gln Val Asn Ser Leu Pro Gly Ser Ile 1155 1160 1165 aac aag gca ggg gag tat att gaa gcc agt tac atg aac ctg cag aga 3611 Asn Lys Ala Gly Glu Tyr Ile Glu Ala Ser Tyr Met Asn Leu Gln Arg 1170 1175 1180 1185 cca tac aca gtg gcc att gct ggg tat gcc ctg gcc ctg atg aac aaa 3659 Pro Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Leu Met Asn Lys 1190 1195 1200 ctg gag gaa cct tac ctc ggc aag ttt ctg aac aca gcc aaa gat cgg 3707 Leu Glu Glu Pro Tyr Leu Gly Lys Phe Leu Asn Thr Ala Lys Asp Arg 1205 1210 1215 aac cgc tgg gag gag cct gac cag cag ctc tac aac gta gag gcc aca 3755 Asn Arg Trp Glu Glu Pro Asp Gln Gln Leu Tyr Asn Val Glu Ala Thr 1220 1225 1230 tcc tac gcc ctc ctg gcc ctg ctg ctg ctg aaa gac ttt gac tct gtg 3803 Ser Tyr Ala Leu Leu Ala Leu Leu Leu Leu Lys Asp Phe Asp Ser Val 1235 1240 1245 ccc cct gta gtg cgc tgg ctc aat gag caa aga tac tac gga ggc ggc 3851 Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly Gly 1250 1255 1260 1265 tat ggc tcc acc cag gct acc ttc atg gta ttc caa gcc ttg gcc caa 3899 Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala Gln 1270 1275 1280 tat caa aca gat gtc cct gac cat aag gac ttg aac atg gat gtg tcc 3947 Tyr Gln Thr Asp Val Pro Asp His Lys Asp Leu Asn Met Asp Val Ser 1285 1290 1295 ttc cac ctc ccc agc cgt agc tct gca acc acg ttt cgc ctg ctc tgg 3995 Phe His Leu Pro Ser Arg Ser Ser Ala Thr Thr Phe Arg Leu Leu Trp 1300 1305 1310 gaa aat ggc aac ctc ctg cga tcg gaa gag acc aag caa aat gag gcc 4043 Glu Asn Gly Asn Leu Leu Arg Ser Glu Glu Thr Lys Gln Asn Glu Ala 1315 1320 1325 ttc tct cta aca gcc aaa gga aaa ggc cga ggc aca ttg tcg gtg gtg 4091 Phe Ser Leu Thr Ala Lys Gly Lys Gly Arg Gly Thr Leu Ser Val Val 1330 1335 1340 1345 gca gtg tat cat gcc aaa ctc aaa agc aaa gtc acc tgc aag aag ttt 4139 Ala Val Tyr His Ala Lys Leu Lys Ser Lys Val Thr Cys Lys Lys Phe 1350 1355 1360 gac ctc agg gtc agc ata aga cca gcc cct gag aca gcc aag aag ccc 4187 Asp Leu Arg Val Ser Ile Arg Pro Ala Pro Glu Thr Ala Lys Lys Pro 1365 1370 1375 gag gaa gcc aag aat acc atg ttc ctt gaa atc tgc acc aag tac ttg 4235 Glu Glu Ala Lys Asn Thr Met Phe Leu Glu Ile Cys Thr Lys Tyr Leu 1380 1385 1390 gga gat gtg gac gcc act atg tcc atc ctg gac atc tcc atg atg act 4283 Gly Asp Val Asp Ala Thr Met Ser Ile Leu Asp Ile Ser Met Met Thr 1395 1400 1405 ggc ttt gct cca gac aca aag gac ctg gaa ctg ctg gcc tct gga gta 4331 Gly Phe Ala Pro Asp Thr Lys Asp Leu Glu Leu Leu Ala Ser Gly Val 1410 1415 1420 1425 gat aga tac atc tcc aag tac gag atg aac aaa gcc ttc tcc aac aag 4379 Asp Arg Tyr Ile Ser Lys Tyr Glu Met Asn Lys Ala Phe Ser Asn Lys 1430 1435 1440 aac acc ctc atc atc tac cta gaa aag att tca cac acc gaa gaa gac 4427 Asn Thr Leu Ile Ile Tyr Leu Glu Lys Ile Ser His Thr Glu Glu Asp 1445 1450 1455 tgc ctg acc ttc aaa gtt cac cag tac ttt aat gtg gga ctt atc cag 4475 Cys Leu Thr Phe Lys Val His Gln Tyr Phe Asn Val Gly Leu Ile Gln 1460 1465 1470 ccc ggg tcg gtc aag gtc tac tcc tat tac aac ctc gag gaa tca tgc 4523 Pro Gly Ser Val Lys Val Tyr Ser Tyr Tyr Asn Leu Glu Glu Ser Cys 1475 1480 1485 acc cgg ttc tat cat cca gag aag gac gat ggg atg ctc agc aag ctg 4571 Thr Arg Phe Tyr His Pro Glu Lys Asp Asp Gly Met Leu Ser Lys Leu 1490 1495 1500 1505 tgc cac agt gaa atg tgc cgg tgt gct gaa gag aac tgc ttc atg caa 4619 Cys His Ser Glu Met Cys Arg Cys Ala Glu Glu Asn Cys Phe Met Gln 1510 1515 1520 cag tca cag gag aag atc aac ctg aat gtc cgg cta gac aag gct tgt 4667 Gln Ser Gln Glu Lys Ile Asn Leu Asn Val Arg Leu Asp Lys Ala Cys 1525 1530 1535 gag ccc gga gtc gac tat gtg tac aag acc gag cta acc aac ata aag 4715 Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Glu Leu Thr Asn Ile Lys 1540 1545 1550 ctg ttg gat gat ttt gat gag tac acc atg acc atc cag cag gtc atc 4763 Leu Leu Asp Asp Phe Asp Glu Tyr Thr Met Thr Ile Gln Gln Val Ile 1555 1560 1565 aag tca ggc tca gat gag gtg cag gca ggg cag caa cgc aag ttc atc 4811 Lys Ser Gly Ser Asp Glu Val Gln Ala Gly Gln Gln Arg Lys Phe Ile 1570 1575 1580 1585 agc cac atc aag tgc aga aac gcc ctg aag ctg cag aaa ggg aag aag 4859 Ser His Ile Lys Cys Arg Asn Ala Leu Lys Leu Gln Lys Gly Lys Lys 1590 1595 1600 tac ctc atg tgg ggc ctc tcc tct gac ctc tgg gga gaa aag ccc aac 4907 Tyr Leu Met Trp Gly Leu Ser Ser Asp Leu Trp Gly Glu Lys Pro Asn 1605 1610 1615 acc agc tac atc att ggg aag gac acg tgg gtg gag cac tgg cct gag 4955 Thr Ser Tyr Ile Ile Gly Lys Asp Thr Trp Val Glu His Trp Pro Glu 1620 1625 1630 gca gaa gaa tgc cag gat cag aag tac cag aaa cag tgc gaa gaa ctt 5003 Ala Glu Glu Cys Gln Asp Gln Lys Tyr Gln Lys Gln Cys Glu Glu Leu 1635 1640 1645 ggg gca ttc aca gaa tct atg gtg gtt tat ggt tgt ccc aac tga 5048 Gly Ala Phe Thr Glu Ser Met Val Val Tyr Gly Cys Pro Asn 1650 1655 1660 ctacagccca gccctctaat aaagcttcag ttgtatttc 5087 <210> SEQ ID NO 112 <211> LENGTH: 494 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <400> SEQUENCE: 112 gctggcttca aacagcccag ctctgcctat gctgccttca acaaccggcc ccccagcacc 60 tgggtagcgg gttgtcagct ctgtcccctc tgcctcaaca tccacgtgag caaagcctga 120 ttccccacca gtggtggtct ggcctctctc tgtcaaggct gcagggactg aatgagcctt 180 agagtccttt aagcaccagc tttatgcggc tttgaaatta aaaatccata actgagggct 240 ctgcaccagg ccctctctgg tcattggtgg gtgaagatgt caatctatct actaaaacca 300 atcgagtctc agctggtgtt cctataactc cgccccagct gacagcctac gtggtcaagg 360 tcttctctct agctgccaac ctcatcgcca tcgactctca cgtcctgtgt ggggctgtta 420 aatggttgat tctggagaaa cagaagccgg atggtgtctt tcaggaggat gggcccgtga 480 ttcaccaaga aatg 494 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 113 gctggtccca tagtgaagga 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 114 cagcagtagc actagtagct 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 115 gggagctggc caacagcagc 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 116 gctcctgtca acactgtctt 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 117 tactggctgg aatcttgatg 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 118 ccccgaagtt tgccaccact 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 119 gatgacgact gtcttgccca 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 120 gggcagcacg tattccttca 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 121 ggccagagaa atcttcttat 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 122 ggatcccact gcgctctgcc 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 123 aatttgggtg tcttggtgaa 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 124 ttgtgcatag tgctgtaggg 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 125 cgcaggtgga agttgacatt 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 126 cttccccttg ttcataacca 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 127 ggtgtagtaa gccaccaggc 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 128 cttgtccaca gccactagcc 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 129 agttcttccc actgcctggg 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 130 gtgtactgac cagctttgtc 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 131 ctcgccctgg gtgatgaggc 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 132 tcactcctgg ccaggcccag 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 133 tgggaagtgg cttctagaga 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 134 ttcaactctt ctatggtcca 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 135 tggaatcttt gagaaagatg 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 136 caggtggtga tggaatcttt 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 137 acaagctcac tgccagaatc 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 138 ttgaagagca cagctctgat 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 139 ggccatgctg cagaaggctg 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 140 tccttctggc acgaccttca 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 141 tctgtgtctg gcacttggtc 20 <210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 142 gtgtttcagc cgctccccgt 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 143 tcacgatcag gtgtttcagc 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 144 gtgtcatgcc aatcatgttc 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 145 ccctttcttg atgagctcca 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 146 gttgaaggca gcataggcag 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 147 gctgtcagcc aggtgctggg 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 148 cttctgtttc tccagaatca 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 149 tcctcctgaa agacaccatc 20 <210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 150 ttcctgcagt gcgatgagga 20 <210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 151 ttgacctgcc cctcacagat 20 <210> SEQ ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 152 cctgccttgt tgatgctccc 20 <210> SEQ ID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 153 gtatggtctc tgcaggttca 20 <210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 154 agggcatacc cagcaatggc 20 <210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 155 ccagtttgtt catcagggcc 20 <210> SEQ ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 156 gtaaggttcc tccagtttgt 20 <210> SEQ ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 157 tcctcccagc ggttccgatc 20 <210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 158 ttcagcagca gcagggccag 20 <210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 159 ggcacagagt caaagtcttt 20 <210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 160 gaataccatg aaggtagcct 20 <210> SEQ ID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 161 ccaaggcttg gaataccatg 20 <210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 162 cacatccatg ttcaagtcct 20 <210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 163 tgaccctgag gtcaaacttc 20 <210> SEQ ID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 164 ggcttcttgg ctgtctcagg 20 <210> SEQ ID NO 165 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 165 gatgtccagg atggacatag 20 <210> SEQ ID NO 166 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 166 aaagccagtc atcatggaga 20 <210> SEQ ID NO 167 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 167 tcttgttgga gaaggctttg 20 <210> SEQ ID NO 168 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 168 atcttttcta ggtagatgat 20 <210> SEQ ID NO 169 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 169 gagtagacct tgaccgaccc 20 <210> SEQ ID NO 170 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 170 atgatagaac cgggtgcatg 20 <210> SEQ ID NO 171 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 171 ggctcacaag ccttgtctag 20 <210> SEQ ID NO 172 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 172 gcctgacttg atgacctgct 20 <210> SEQ ID NO 173 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 173 cctgcctgca cctcatctga 20 <210> SEQ ID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 174 gaggccccac atgaggtact 20 <210> SEQ ID NO 175 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 175 gggcttttct ccccagaggt 20 <210> SEQ ID NO 176 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 176 cttcccaatg atgtagctgg 20 <210> SEQ ID NO 177 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 177 cagtgctcca cccacgtgtc 20 <210> SEQ ID NO 178 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 178 ggctgtagtc agttgggaca 20 <210> SEQ ID NO 179 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 179 aaatacaact gaagctttat 20 

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding complement component C3, wherein said compound specifically hybridizes with said nucleic acid molecule encoding complement component C3 and inhibits the expression of complement component C3.
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 19, 20, 21, 23, 24, 26, 29, 30, 31, 32, 34, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 83, 84, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 100, 107, 33, 113, 114, 115, 116, 117, 118, 120, 121, 123, 124, 125, 126, 127, 129, 130, 131, 132, 133, 135, 136, 138, 140, 141, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 157, 158, 159, 160, 161, 163, 164, 166, 169, 174, 175, 176, 177, 178 or
 179. 4. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 5. The compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 6. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 7. The compound of claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 9. The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding complement component C3.
 12. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 13. The composition of claim 12 further comprising a colloidal dispersion system.
 14. The composition of claim 12 wherein the compound is an antisense oligonucleotide.
 15. A method of inhibiting the expression of complement component C3 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of complement component C3 is inhibited.
 16. A method of treating an animal having a disease or condition associated with complement component C3 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of complement component C3 is inhibited.
 17. The method of claim 16 wherein the disease or condition is an autoimmune disorder.
 18. The method of claim 17 wherein the autoimmune disorder is multiple sclerosis.
 19. The method of claim 18 wherein the disease or condition is an infection.
 20. The method of claim 16 wherein the disease or condition is atherosclerosis. 